Osteocytes play a critical role in translating mechanical loading into signals that regulate mineralization, bone formation and resorption. Our preliminary data demonstrate that a member of the Sibling family of matrix proteins, MEPE/osteogenic factor 45, is highly expressed in alveolar bone osteocytes. The expression of MEPE increases in response to mechanical stimulation several fold in vivo in the tooth movement model. This secreted protein is thought to function in the osteocyte-lacunar-canalicular mineralization process and in the local modification of the osteocyte microenvironment. Thus, MEPE is a good candidate for osteocyte signaling during mechanical loading. The proposed studies are based on the hypothesis that specific region(s) of MEPE gene control its expression in osteocytes in response to mechanical stimulation. The major focus of this application is to identify the cis-regulatory regions in the MEPE gene that control response of osteocytes to mechanical loading in vivo. To achieve this goal we will create MEPE transgenic mice with a 10-20 kb promoter region. This region will be defined using VISTA program, and comparing conserved regions in mouse MEPE gene with other species. The promoter region defined in transgenic mice will be driven by the green florescent protein (GFP) as a reporter gene. In vivo tooth movement mouse model developed in our laboratory will be used to test the responsiveness of MEPE/GFP transgene to mechanical loading and to correlate the regulation of transgene and the MEPE gene. This will be a novel approach to study a complex signaling between bone cells during mechanical loading. We will also use an in vitro cell culture system using stably transfected cells in 2T3 mineralized cultures stimulated by fluid flow to carry out additional screening of candidate MEPE gene enhancers and modules. Mechanically responsive and osteocyte specific constructs determined in this study will be utilized in future studies to generate new transgenic mouse models. This approach should enhance our understanding of the complex signaling pathways involved in bone response to mechanical loading. [unreadable] [unreadable]